Polyrotaxane Composed of Poly-l-lactide and α ... - ACS Publications

Tomohiro Seki , Keigo Abe , Yuya Egawa , Ryotaro Miki , Kazuhiko Juni , and Toshinobu Seki. Molecular Pharmaceutics 2016 13 (11), 3807-3815. Abstract ...
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Biomacromolecules 2009, 10, 2261–2267

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Polyrotaxane Composed of Poly-L-lactide and r-Cyclodextrin Exhibiting Protease-Triggered Hydrolysis Yuichi Ohya,*,†,‡ Seigo Takamido,† Koji Nagahama,†,⊥ Tatsuro Ouchi,†,‡ Ryo Katoono,§,| and Nobuhiko Yui*,§,| Department of Chemistry and Materials Engineering, Faculty of Chemistry, Materials and Bioengineering, and High Technology Research Center, Kansai University, 3-3-35 Yamate, Suita, Osaka 564-8680, Japan, School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi, Ishikawa 923-1292, Japan, and JST CREST, 5 Sanbancho, Chiyoda, Tokyo 102-0075, Japan Received April 17, 2009; Revised Manuscript Received June 2, 2009

Biodegradable polyrotaxanes (PRXs) were synthesized from bis-amino-terminated poly(L-lactide) (PLLA) and R-cyclodextrin (R-CD), combined with capping by bulky end groups at the amino-termini of PLLA through enzymatically cleavable peptide linkages. The crystalline structure of the PRXs in the solid state was investigated by wide-angle X-ray diffraction (WAXRD), and the results suggested that PRX forms a column-type crystalline structure. Hydrolysis of ester bonds of the PLLA in PRX was prevented due to the supramolecular structure. However, in the presence of a protease (papain), the hydrolysis of PLLA in PRX was induced. The removal of the bulky end groups by the protease acted as a trigger for the release of R-CD and allowed hydrolysis of the PLLA ester bonds. Such unique hydrolysis behavior, that is, proteinase-triggered degradation of a polyester, was achieved by the combination of the supramolecular architecture, biodegradable PLLA, and enzymatically cleavable end groups.

Introduction Biodegradable synthetic polymers have attracted much interest not only for ecological and environmental situations but also for biomedical and pharmaceutical applications.1-4 In the biomedical field, biocompatible polymers that exhibit a suitable degradation profile in the body after certain periods of time are required for implantable biomedical materials for drug delivery systems (DDS) and tissue engineering. Poly(L-lactide) (PLLA) has frequently been used in implantable carriers for DDS and surgical repair materials because of its biodegradability, biocompatibility, high mechanical strength, and good shaping and molding properties.5-9 The control of the degradation behavior of PLLA-based materials has been extensively investigated in the past two decades.10-22 Implantable biomedical materials, such as drug delivery carriers and cellular scaffolds for tissue engineering, often must be readily decomposed after use for a given purpose. Therefore, the development of biodegradable materials having stimuliresponsive or -triggered decomposition properties is an important challenge for innovation of implantable biomedical materials. Although simple acceleration of the degradation of PLLA-based materials could be achieved,10-22 specific stimuli-responsive degradation or rapid degradation at a desired time was not achieved. Pseudopolyrotaxane and polyrotaxane, composed of linear polymers and cyclic compounds, have attracted much attention * To whom correspondence should be addressed. Fax +81-6-6368-0818 (Y.O.); +81-76-151-1645 (N.Y.). E-mail: [email protected] (Y.O.); [email protected] (N.Y.). † Department of Chemistry and Materials Engineering, Kansai University. ‡ High Technology Research Center, Kansai University. § Japan Advanced Institute of Science and Technology. | JST CREST. ⊥ Present address: Department of Nanobiochemistry, Frontiers of Innovative Research in Science and Technology (FIRST), Konan University, 7-1-20 Minatojima-Minamimachi, Chuo-ku, Kobe, 650-0047, Japan.

due to their unique supramolecular structures and their corresponding functions.22-28 Harada et al. and Tonelli et al. have reported the synthesis of pseudopolyrotaxanes consisting of polyesters, such as poly(ε-caprolactone) (PCL)29 and poly(3hydroxylbutyrate) (PHB),30-32 and R-cyclodextrins (R-CDs). Recently, we have also found that linear PLLA and PLLA-PEGPLLA triblock copolymer could form pseudopolyrotaxanes with R-CDs.33,34 CDs have been widely employed as substraterecognition moieties in enzyme models, such as serine proteases, papain, and esterase, because of their cavitand nature and hydroxyl groups around the rims of the cavities.35-39 Bender and Komiyama et al. reported that CDs accelerate the hydrolysis of activated esters, such as p-nitrophenyl acetate.40-42 Recently, Harada et al. reported that R-, β-, and γ-CDs selectively form inclusion complexes with lactones, such as δ-valerolactone, β-butyrolactone, and ε-caprolactone, and the hydrolysis of these lactones is promoted or suppressed according to the size of the CDs.43 In this study, we synthesized a biodegradable polyrotaxane composed of PLLA and R-CDs (LA-PRX). The dethreading of the R-CDs was prevented by blocking the PLLA chain with bulky end groups through enzymatically cleavable peptide linkages. Here we report on the protease-triggered degradation behavior of LA-PRX. The obtained LA-PRX was not degraded in the absence of papain but exhibited a higher degradation rate in response to papain, which catalyzed enzymatic hydrolysis of the peptide bonds.

Experimental Section Materials. L-Lactide (L-LA) was purchased from Purac Biochem BV (Gorinchem, The Netherlands) and used without further treatment. R-CD, tin 2-ethylhexanoate (Sn(oct)2), papain (EC 3,4,22,2) from papaya latex (Activity: 1153 unit/mL protein), and other chemicals were purchased from Wako Pure Chemical Co. Benzyloxycarbonyl-Lphenylalanine succinimide ester (Z-L-Phe-OSu) was purchased from

10.1021/bm900440v CCC: $40.75  2009 American Chemical Society Published on Web 07/02/2009

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Scheme 1. Synthetic Route of Polyrotaxane Composed of Poly(L-lactide) and R-CDs

Kokusan Chemical Co. Organic solvents were purified by the usual distillation methods. The other synthetic reagents were used as received without further purification. Synthesis of Bis-hydroxyl-Terminated Polylactide. Bis-hydroxylterminated PLLA was synthesized through ring-opening polymerization of L-LA in bulk using triethylene glycol (TEG) as a bifunctional initiator and Sn(oct)2 as a catalyst,34 as shown in Scheme 1. In brief, L-LA (4.8 g, 34 mmol) and TEG (100 µL, 0.56 mmol) were placed in a glass tube, and a freshly prepared anhydrous THF solution (100 µL) of Sn(oct)2 (14 mg, 34 µmol) was added to the glass tube under a nitrogen atmosphere. The glass tube was then purged with argon and placed in an oil bath at 150 °C for 2 min and then at 115 °C for 12 h. The reaction mixture was dissolved in chloroform (10 mL) and the resultant solution was poured into diethylether (300 mL) to remove unreacted L-LA. The obtained precipitate was collected by centrifugation and dried under vacuum overnight at room temperature to give bis-hydroxyl-terminated PLLA as a white solid. Number-average molecular weight (Mn) and polydispersity index (Mw/Mn) of the synthesized bis-hydroxyl-terminated PLLA were determined by gel permeation chromatography (GPC; Tosoh GPC-8020 series system; column: TSK-GEL ALPHA-5000 × 2; eluent: DMF; detector: refractive index (RI); standard: polystyrene (PS)). The degree of polymerization of L-LA was determined by 1H NMR spectroscopic analysis using JEOL GSX-400. The specific optical rotation ([R]) of the synthesized bis-hydroxyl-terminated PLLA was measured by dissolving the polymers in chloroform at a concentration of 10 mg/mL and 25 °C ([R]D25) using a polarimeter (HORIBA SEPA-300) at a wavelength of 589 nm. 1H NMR (CDCl3): δ 1.55 (d, CHCH3), 3.50 (s, -OCH2CH2O-), 4.10 [m, CH(CH3)OH], 4.18 (t, -OCH2CH2OCO-), 5.17 (q, CHCH3). Synthesis of Bis-amino-Terminated Polylactide. Bis-aminoterminated PLLA (H2N-PLLA-NH2) was synthesized through coupling reaction between bis-hydroxyl-terminated PLLA with t-butoxycarbonylglycine (Boc-Gly) with subsequent deprotection of the Boc groups, as shown in Scheme 1. Boc-Gly (500 mg, 2.9 mmol) and N,N′-carbonyldiimidazole (CDI; 470 mg, 2.9 mmol) were dissolved in anhydrous CH2Cl2 and the solution was stirred for 4 h at room temperature under a nitrogen atmosphere. Bis-hydroxyl-terminated PLLA (4.0 g, 480 µmol) and 4-dimethylaminopyridine (DMAP; 15 mg, 120 µmol) dissolved in anhydrous CH2Cl2 (7 mL) was added to the reaction mixture. The solution was stirred at room temperature for 6 h under a nitrogen atmosphere. The reaction mixture was poured into diethylether/ methanol (300 mL, 2/1, v/v) to remove unreacted Boc-Gly and imidazole. HBr/CH3COOH (25%; 8.8 mL, 19.4 mmol) was added to the obtained precipitate dissolved in CH2Cl2 (10 mL), and the mixture was stirred for 10 min at 10 °C. Triethylamine (TEA; 6.0 mL, 29.1

mmol) was added and the reaction mixture was stirred for 5 min at 10 °C to neutralize the HBr/CH3COOH. The reaction mixture was filtered to remove reaction byproduct salts. The obtained filtrate was evaporated and rinsed with methanol (30 mL × 3 times). The product was dried under vacuum overnight at room temperature to give H2N-PLLA-NH2 as a white solid. The degree of substitution of Boc-Gly and deprotection of Boc groups were estimated by 1H NMR spectroscopy to be almost 100%. 1H NMR (CDCl3): δ 1.55 (d, CHCH3), 3.50 (s, -OCH2CH2O-), 3.60 (t, -OCOCH2NH2), 4.18 (t, -OCH2CH2OCO-), 5.17 (q, CHCH3). Preparation of Pseudopolyrotaxanes. Pseudopolyrotaxanes composed of H2N-PLLA-NH2 and R-CD (LA-pPRX) were synthesized in the bulk state using the vacuum melting technique at 170 °C.39 Under a nitrogen atmosphere, H2N-PLLA-NH2 (3.0 g, 360 µmol) and R-CD (23.1 g, 23.7 mmol) were placed in a glass tube. The tube was purged with argon gas and then placed under vacuum at reduced pressure. The sealed tube was heated in an oil bath at 170 °C for 1 h with vigorous stirring. Next, the glass flask was cooled to 60 °C and the reaction was continuously stirred for 1 h under reduced pressure. This heating and cooling cycle was repeated 12 times. The reaction mixture was washed with large amounts of water and acetone to remove uncomplexed R-CD and H2N-PLLA-NH2. The insoluble product in acetone was collected by centrifugation and dried under vacuum at room temperature for 24 h to give LA-pPRX as a white solid (yield ) 41.3%). The average number of R-CDs threaded onto a H2N-PLLA-NH2 molecule for LApPRX was determined by 1H NMR spectroscopy after hydrolysis in 10% NaOD/D2O. 1H NMR (DMSO-d6): δ 1.48-1.85 (m, CHCH3), 3.11-3.21 [m, C(2)H and C(4)H], 3.34 (s, -OCH2CH2O-), 3.64-3.74 [m, C(3)H, C(6)H and C(5)H], 4.35 (t, -OCH2CH2OCO-), 4.60 [s, O(6)H], 4.80 [s, C(1)H], 5.17 (q, CHCH3), 5.06 [s, O(3)H], 5.25 [s, O(2)H]. Synthesis of Polyrotaxanes. Polyrotaxane composed of a PLLA chain, R-CD, and end-capping groups (LA-PRX) was synthesized through a coupling reaction between H2N-PLLA-NH2 in the LA-pPRX with Z-L-Phe succinimide ester (Z-L-Phe-OSu), as shown in Scheme 1. LA-pPRX (5.0 g, 147 µmol) and Z-L-Phe-OSu (2.2 g, 5.6 mmol) were dissolved in anhydrous DMSO saturated with R-CD (20 mL) and stirred at room temperature for 48 h. The reaction mixture was poured into a large amount of diethylether and the resultant precipitate was collected by centrifugation. The obtained precipitate was thoroughly rinsed with large amounts of acetone and water to remove free H2NPLLA-NH2, R-CD, and Z-L-Phe-OSu. The product was dried under vacuum overnight at room temperature to give LA-PRX as a white solid (yield ) 24.1%). The average number of R-CDs threaded onto a PLLA molecule was determined by 1H NMR spectroscopy after hydrolysis in 10% NaOD/D2O. Purity of the synthesized LA-PRX was

Protease-Triggered Degradation of PLLA Polyrotaxane

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Figure 1. (A) 1H NMR spectrum of LA-PRX in DMSO-d6. (B) GPC profiles of (a) LA-PRX, (b) H2N-PLLA-NH2, and (c) R-CD.

estimated by GPC measurement using a Tosoh HLC-8120 GPC system (column: TSKgel G-5000HHR + TSKgel G-3000HHR; flow rate: 0.8 mL/min; detector: RI and optical rotation; standard: PEG; eluent: DMSO). 1H NMR (DMSO-d6): δ 1.48-1.85 (m, CHCH3), 3.11-3.21 [m, C(2)H and C(4)H], 3.34 (s, -OCH2CH2O-), 3.64-3.74 [m, C(3)H, C(6)H and C(5)H], 4.35 (t, -OCH2CH2OCO-), 4.60 [s, O(6)H], 4.80 [s, C(1)H], 5.17 (q, CHCH3), 5.06 [s, O(3)H], 5.25 [s, O(2)H], 7.40-7.55 (m, aromatic protons of Z-L-Phe). Measurements. Thermal properties of H2N-PLLA-NH2, LA-pPRX, and LA-PRX were analyzed by a differential scanning calorimetor (DSC; Shimadzu DSC-60, TA-60WS) measurements under a flow of nitrogen gas (40 cm3/min). The temperature examined ranged from 0 to 200 °C with a heating rate of 10 °C/min. Crystalline structures of the H2N-PLLA-NH2, LA-pPRX, and LA-PRX were analyzed by a wide-angle X-ray diffractmeter [WAXRD; Mac Science Co., M18XHF22SRA instrument with Cu Ka source (λ ) 1.54 Å)] at 25 °C. In Vitro Hydrolytic Degradation. Hydrolytic degradation behaviors of the H2N-PLLA-NH2, LA-pPRX, and LA-PRX were investigated by mass loss and molecular weight reduction. Samples were molded in tablet dies (P/N 202-32010, Shimadzu Co.) by a press instrument (SSP-10A, Shimadzu Co.) to give flat disks about 18 mm in diameter × 0.45 mm in thickness. The disks were weighed after thorough drying (W0) and incubated in 2 mL of 6.7 × 10-2 M KH2PO4-Na2HPO4 buffer (PBS; pH ) 7.4, ionic strength ) 0.14) at 37 °C for 56 days. At predetermined time periods, the disks were washed with pure water, dried under vacuum, and then weighed (Wt). The mass remaining was determined as follows

mass remaining(%) ) Wt /W0 × 100 The ester bond hydrolysis (molecular weight reduction) of H2NPLLA-NH2, PLLA in LA-pPRX, and PLLA in the LA-PRX was investigated in PBS at 37 °C. At predetermined time periods, the disks of the polymers were washed with pure water and dried under vacuum. The Mn of the PLLA chain after hydrolysis was determined by GPC measurement. In the cases of LA-pPRX and LA-PRX, the degraded disks were dissolved in DMF and the solutions were sonicated at 60 °C to dissociate the R-CDs and PLLA chain. The PLLA component was extracted from the resultant mixtures by washing with acetone and drying under vacuum. After these treatments, Mn of the PLLAs after hydrolysis was determined by GPC measurement. The molecular weight remaining of the PLLA chain was calculated from the initial molecular weight (M0) and the molecular weight after hydrolysis (Mt)

molecular weight remaining(%) ) Mt /M0 × 100

WAXRD measurement of the polymers was carried out after drying to analyze changes in the crystalline structure during the hydrolytic degradation process. In Vitro Enzymatic Degradation. Degradation behaviors of the H2N-PLLA-NH2, LA-pPRX, and LA-PRX in the presence or absence of papain were investigated by a similar method as described above using 2 mL of PBS (pH ) 7.4, ionic strength ) 0.18) containing papain (87 mL, 100 unit), ethylenediaminetetraacetic acid (EDTA; 2 µmol/ mL), and 2-mercaptoethanol (10 µmol/mL) at 37 °C for 56 days. Mass remaining (%) and molecular weight remaining (%) were estimated by the same methods with hydrolytic degradation tests, as described above.

Results and Discussion Characterization of the Polyrotaxanes. Bis-hydroxylterminated PLLA having a molecular weight of 8200 Da and a narrow molecular weight distribution (1.12) was obtained by ring-opening polymerization of L-LA in the presence of TEG. H2N-PLLA-NH2 was obtained by the coupling reaction of bishydroxyl-terminated PLLA with Boc-Gly and subsequent deprotection. The specific optical rotation, [R]D25, of H2N-PLLANH2 in CHCl3 was -137°. This value is very close to that of pure PLLA.44 Because PLLA is insoluble in water, which is a common solvent for preparing pPRX composed of PEG and R-CDs, the preparation of a LA-pPRX composed of H2NPLLA-NH2 and R-CDs was carried out in the bulk using vacuum melting at 170 °C, according to a procedure previously reported.34 The results from the preparation of LA-pPRX are listed in Table S2 (Supporting Information). The average number of R-CD molecules threaded onto a PLLA chain was estimated to be 26 from the 1H NMR integral ratio of the C(1)H signal assigned to R-CD and the CH3 signal assigned to lactic acid of LA-pPRX in DMSO-d6. Considering the stretched length of the lactide unit and the depth of the R-CD cavity, one R-CD molecule should be equivalent to two lactic acid units (one lactide unit), as reported for the case of PEG.23,24 Estimating from these theoretical parameters, the threaded R-CD (%) on H2N-PLLA-NH2 was 45% of theoretical maximum. The Mn of LA-pPRX was estimated to be 33600 Da from the following equation:

Mn(LA-pPRX) ) (Mn of PLLA) + (total molecular weight of threaded R-CDs onto a PLLA) LA-PRX was synthesized by coupling reaction of both amino termini of H2N-PLLA-NH2 in LA-pPRX with Z-L-Phe-OSu in

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Figure 2. DSC curves of (a) H2N-PLLA-NH2, (b) LA-pPRX, and (c) LA-PRX.

R-CD saturated DMSO. Figure 1A and Figure S1 show the 1H NMR spectra of the obtained LA-PRX and H2N-PLLA-NH2, respectively. In LA-PRX, the methyl protons showed quite different resonance peaks at 1.7 ppm in comparison with the corresponding signals for H2N-PLLA-NH2. The signals for the methyl protons of the lactide unit shifted slightly to a lower magnetic field, and the peak was split into two major and two minor peaks. These results showed good agreements with our previous report for LA-pPRX.34 Figure 1B shows the GPC curves of LA-PRX, H2N-PLLA-NH2,and R-CD. A single peak having positive optical rotation was observed at around 36 min for LA-PRX with a retention time significantly shorter than that of the corresponding H2N-PLLA-NH2 and R-CD, suggesting that R-CDs were actually threaded onto PLLA chains and free H2N-PLLA-NH2 and R-CDs did not contaminate the LA-PRX. Moreover, this GPC system could detect the optical rotation of the compounds subjected to it, that is, peaks for compounds with negative optical rotation appear in the negative territory and those for compounds with positive optical rotation appear in the positive territory. Thus, peaks for PLLA and R-CD were observed in the negative and the positive territory, respectively, whereas the peak for LA-PRX was observed in the positive territory. These results provide us clear evidence for the physically interlocked supramolecular structure of LA-PRX, where the PLLA chain (optical-rotationally negative) is covered with R-CD (optical-rotationally positive) molecules. The number of R-CDs threaded onto a PLLA chain and threaded R-CD (%) were determined to be 24 and 43% by the same methods as LA-pPRX. Crystalline Structure of the Polyrotaxanes. The thermal properties of the obtained LA-PRX, corresponding LA-pPRX and H2N-PLLA-NH2 in the solid state were investigated by DSC. Figure 2 shows the DSC curves for LA-PRX, LA-pPRX, and H2N-PLLA-NH2. The thermogram of H2N-PLLA-NH2 exhibited a sharp endothermal peak at 160 °C attributed to the melting of PLLA crystals. On the other hand, no obvious melting peak was observed in either LA-PRX or LA-pPRX thermogram. These results indicate that crystallization of PLLA did not occur in the bulk LA-PRX and LA-pPRX. The WAXRD patterns of H2N-PLLA-NH2, LA-PRX, and LA-pPRX are shown in Figure 3. The presence of a large number of reflections in the diffraction patterns of the samples suggests their crystallinity. However, the diffractograms of both the LA-pPRX and LA-PRX showed a quite different pattern from that of the H2N-PLLA-NH2. Characteristic peaks corresponding to the typical PLLA crystal were observed at 2θ ) 17° and 19° for H2N-PLLA-NH2. This result is coincident with

Ohya et al.

Figure 3. Wide-angle X-ray diffraction patterns of (a) H2N-PLLA-NH2, (b) LA-pPRX, and (c) LA-PRX.

Figure 4. Plots of mass remaining vs time for LA-PRX (9) and H2NPLLA-NH2 (b) in PBS at 37 °C.

the DSC thermogram described above. Such diffraction peaks were not observed for LA-pPRX and LA-PRX, however, a weak but obvious peak appeared at 2θ ) 20° corresponding to the R-CD columnar crystalline structure.23,24 Hydrolytic Degradation Profile of the Polyrotaxanes. The PLLA component of LA-PRX is biodegradable. In order to investigate the degradation behavior of the PLLA chain in the LA-PRX, tablets of H2N-PLLA-NH2 and LA-PRX were soaked in PBS and the mass loss was monitored at 37 °C as a function of time. Figure 4 shows the time course of mass remaining of H2N-PLLA-NH2 and LA-PRX. The mass loss of LA-PRX was minimal for at least for 56 days, whereas the mass of H2NPLLA-NH2 decreased gradually to about 85% after 56 days incubation in PBS. These results indicate that almost no hydrolysis of the PLLA chain in LA-PRX occurred due to the threading of R-CDs. Interestingly, although the threaded R-CD (%) on the H2N-PLLA-NH2 chain was only 44.8% and PLLA in LA-PRX was not crystallized, the hydrolysis of the PLLA chain in LA-PRX was nearly prevented. WAXRD patterns of dry LA-PRX samples after 28 and 56 days were also analyzed, as shown in Figure S2 (Supporting Information). These WAXRD patterns were almost the same as the original LA-PRX sample, and a peak at 2θ ) 20° was also observed clearly in both cases, meaning that the polyrotaxane structure was maintained intact after exposure to PBS for 56 days. Thus, it is concluded that R-CD threaded onto PLLA chains can prevent ester hydrolysis of the PLLA molecule. Enzymatic Degradation Profile of the Polyrotaxanes. We investigated the effects of dissociation of the supramolecular

Protease-Triggered Degradation of PLLA Polyrotaxane

Figure 5. Plots of mass remaining vs time for LA-PRX in the presence of papain (9) and LA-pPRX in the absence of papain (b) in PBS at 37 °C.

Figure 6. Plots of molecular weight remaining vs time for LA-PRX in the presence of papain (9), LA-pPRX in the absence of papain (0), H2N-PLLA-NH2 in the presence of papain (b), and H2N-PLLA-NH2 in the absence of papain (O) in PBS at 37 °C.

polyrotaxane structure by enzymatic cleavage of the peptide linkages of the bulky end groups on the hydrolysis behavior of LA-PRX in the presence of protease papain, which can cleave the Phe-Gly peptide linkages. Figure 5 shows the time course of mass remaining of LA-PRX in the presence of papain in PBS at 37 °C. As a control, mass remaining of LA-pPRX without end-capping groups in PBS at 37 °C (without papain) was also investigated. Drastic decreases in mass were observed for LA-PRX and LA-pPRX. The mass decrease rate for LApPRX was faster than that for LA-PRX. The mass losses for the LA-PRX and LA-pPRX were reached to about 50% after 21 and 7 days, respectively. After these periods, the mass decreases for LA-PRX and LA-pPRX became relatively slow. These LA-PRX and LA-pPRX contain about 75 wt % of R-CDs. So, the mass losses observed here should be mainly due to the release of R-CDs. Figure 6 shows the time course of molecular weight remaining of H2N-PLLA-NH2 and PLLA chains in LA-PRX and LA-pPRX. The decreases in the molecular weight of H2N-PLLA-NH2 were gradual during the incubation time, and the behaviors were almost the same in the absence and presence of papain. These results mean that papain has no influence on the degradation behavior of the H2NPLLA-NH2. Interestingly, the molecular weight of the PLLA chain in LA-PRX was almost constant for the first 7 days and

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Figure 7. Changes in wide-angle X-ray diffraction patterns for LAPRX during degradation test in the presence of papain in PBS at 37 °C.

then drastically decreased within 28 days. After that, the degradation rate of PLLA chain became slower. The release of R-CD was not taken into account in the molecular weight decrease data. Instead, the molecular weight reduction is a net value for the PLLA chain. These results indicate that the mass loss observed in the first 7 days was only due to the release of R-CD triggered by enzymatic cleavage of the peptide linkages catalyzed with papain. A time lag (ca. 7 days) between mass loss and molecular weight reduction of PLLA was observed. In contrast, drastic molecular weight reduction of the PLLA chain was observed from day 2 in the case of LA-pPRX. Figure S3 (Supporting Information) shows expanded plots for time course molecular weight remaining during the initial 14 days in this degradation test of LA-pPRX. The molecular weight of the PLLA chain in LA-pPRX was constant for the first 2 days. There was a 2 day time lag between the mass loss and the molecular weight reduction, but this was much shorter than that observed for LA-PRX (7 days). After 7 days, molecular weight reduction became slower, and such slowdown phenomena in degradation rates were observed after 21 days in the case of LA-PRX. Crystalline Structure Changes for Polyrotaxane in the Presence of Papain. To investigate the degradation process in detail, WAXRD measurements during the degradation tests were carried out. Figure 7 shows time course WAXRD pattern changes for degradation products of LA-PRX. A peak assigned to the columnar crystalline structure was observed in the patterns for the first 7 days. These results indicate that the columnar crystalline structure was maintained for 7 days. On day 14, the peaks almost disappeared, indicating an amorphous state. Such an amorphous state cannot be observed in the hydrolytic degradation process of typical PLLA. After 21 days, a characteristic peak assigned to normal PLLA crystal appeared. This means the PLLA chain could be crystallized from day 14 to day 21. WAXRD pattern change for LA-pPRX during the degradation test was also shown in Figure S4 (Supporting Information). In Figure S4, peaks assigned to the columnar crystalline structure were observed for the first 2 days, indicating that the sample was in an amorphous state at day 7. Such an amorphous pattern was observed 7 days later (day 14) in the case of LA-PRX (Figure 7). The crystalline peaks assigned to PLLA appeared at day 14 in the case of LA-pPRX. This means the PLLA chain in LA-pPRX could be crystallized from day 7

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Figure 8. Schematic representation for hydrolytic decomposition and degradation of LA-PRX. (a) Threaded R-CDs prevent the hydrolysis of PLLA in LA-PRX. (b) Peptide linkage cleavage at bulky end-capping groups by papain converts LA-PRX into LA-pPRX. (c) Release of R-CDs from LA-pPRX leads amorphous state, exposure of PLLA to water, and the beginning of ester bond hydrolysis in the PLLA chain.

to day 14. These sequential changes in crystalline structure (the columnar crystal state, amorphous state, and PLLA crystal formation) seem to be in good agreement with each stage of the dissociation and degradation process (the release of R-CD, degradation of PLLA chain, and slowdown of the degradation, respectively) observed for LA-PRX and LA-pPRX in Figure 6. It is well-known the fact that degradation rate of crystalline domain of PLLA is slower than that of amorphous PLLA domain. In some cases, it was reported that the apparent increase in the crystallinity of semicrystalline PLLA can be observed during the degradation process and the degradation rate go slow dawn. So, it is reasonable that the increasing PLLA crystalline peak in WAXD pattern was coincident with the decrease in degradation rate. Degradation Mechanism. All the results obtained in the degradation tests for LA-PRX and LA-pPRX suggest that the longer time lag (ca. 7 days) observed for LA-PRX is the period required for enzymatic cleavage of end-capping groups. The degradation rate of LA-PRX was faster than H2N-PLLA-NH2 as well as LA-pPRX. It can be concluded that the temporary amorphous state appearing during the dissociation process for both LA-PRX and LA-pPRX systems is an indispensable factor to induce hydrolysis of the PLLA chain compared to H2NPLLA-NH2, which had a PLLA crystalline structure over all the degradation tests. Special interest should be paid to the fact that the degradation of the PLLA chain in LA-PRX is much greater than that of H2N-PLLA-NH2. The existence of R-CD on PLLA in the pseudopolyrotaxane form is an acceleration factor for degradation of PLLA, although it is an inhibitory factor in polyrotaxane form. Considering these results, the remarkably drastic decrease in the molecular weight of the PLLA chain in LA-PRX was induced during the dissociation process of the polyrotaxane structure. Obviously, the hydrolysis of the ester bonds is not accelerated by papain; papain only acts as a trigger for the hydrolysis. Based on all the results mentioned above, we propose a mechanism for the degradation profile of LA-PRX, as shown in Figure 8: (a) ester hydrolysis of the PLLA chain in LAPRX was prevented by threaded R-CD molecules; (b) peptide bonds between PLLA and bulky end-capping groups were cleaved by catalytic reaction with papain, converting LA-PRX to LA-pPRX; (c) the threaded R-CDs were released and ester hydrolysis of the PLLA began. In this process, a temporary amorphous state due to the release of R-CD molecules is the main factor for faster degradation rates of LA-PRX and LApPRX compared with H2N-PLLA-NH2. After most of the R-CDs were released, the residual PLLA could be crystallized, causing the degradation rate to decrease. As described above, we have achieved protease (papain)triggered degradation of LA-PRX, as a unique type of degradation in biodegradable materials. While it is well-known that esterases can catalyze degradation of polyesters, the degradation mechanism and process described here are completely different.

In this case, the enzyme papain is only a trigger to start the release of R-CDs, which allows subsequent hydrolysis of the PLLA chain. Protease (or peptidase) is the most common and ubiquitous enzyme for animals, and is found in a wide variety of forms. Selective cleavages by proteases for specific amino acids or specific sequences are possible, although the substrate specificity of esterases (for example lipase) is generally not so strict. We may design LA-PRX having specific sequences of end-capping groups, which is expected to exhibit specific protease triggered hydrolysis behavior. In addition, the distribution of protease in the body is quite diverse. Some proteases are expressed in a specific site or for a specific disease such as cancer or thrombosis. This means the LA-PRX system exhibiting protease-triggered degradation behavior developed in this study has great potential and utility for medical applications.

Conclusions Degradation behavior of a supramolecular biodegradable polyrotaxane composed of PLLA and R-CDs (LA-PRX) was investigated in the presence and absence of papain. Ester bond hydrolysis of the PLLA chain in the LA-PRX was nearly prevented because of the R-CD threaded supramolecular structure. Protease (papain)-triggered degradation of LA-PRX was achieved, and the degradation rate after papain treatment was faster than that of normal PLLA. Proteases have a wide variety of specificity for cleavage sites, and specific localization in sites and physiological conditions in the human body. Therefore, the LA-PRX system has great potential and utility for the development of biodegradable medical devices that show specific stimuli-responsive degradation, drug release, or timecontrolled excretion. We postulate that this finding can explore the new paradigm of biodegradable materials based on supramolecular structures for biomedical applications. Acknowledgment. A part of this work was financially supported by a Grant-in-Aid for Scientific Research on Priority Areas (20034055) and Scientific Research B (19300170) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, and a Kansai University Grant-in-Aid for progress of research in graduate courses, 2008. This work was carried out as a part of studies in the High-Tech Research Center Project supported by MEXT, Japan. Supporting Information Available. The characterizations of hydroxyl-terminated PLLA. Changes in WAXRD patterns of LA-pPRX during hydrolytic degradation test. Detailed data for degradation profiles of LA-pPRX and H2N-PLLA-NH2 in PBS at 37 °C. This material is available free of charge via the Internet at http://pubs.acs.org.

Protease-Triggered Degradation of PLLA Polyrotaxane

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